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Modified titanium surface with gelatin nano gold composite increases osteoblast cell biocompatibility
Applied Surface Science 256 (2010) 5882–5887
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Modified titanium surface with gelatin nano gold composite increases osteoblast cell biocompatibility Young-Hee Lee a,1 , Govinda Bhattarai a,1 , Santosh Aryal b , Nan-Hee Lee a , Min-Ho Lee c , Tae-Gun Kim d , Eun-Chung Jhee a , Hak-Yong Kim b , Ho-Keun Yi a,∗ a
Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju, Republic of Korea Department of Bionanosystem Engineering, Chonbuk National University, Jeonju, Republic of Korea Department of Dental Biomaterials, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju, Republic of Korea d Department of Conservative Dentistry, School of Dentistry, Chonbuk National University, Jeonju, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 12 March 2010 Accepted 12 March 2010 Available online 19 March 2010 Keywords: Gelatin nano gold composite Titanium modification Cell signaling Cell adherent molecules Biocompatibility Dental implant
a b s t r a c t This study examined the gelatin nano gold (GnG) composite for surface modification of titanium in addition to insure biocompatibility on dental implants or biomaterials. The GnG composite was constructed by gelatin and hydrogen tetrachloroaurate in presence of reducing agent, sodium borohydrate (NabH4 ). The GnG composite was confirmed by UV–VIS spectroscopy and transmission electron microscopy (TEM). A dipping method was used to modify the titanium surface by GnG composite. Surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). The MC-3T3 E1 cell viability was assessed by trypan blue and the expression of proteins to biocompatibility were analyzed by Western blotting. The GnG composite showed well dispersed character, the strong absorption at 530 nm, roughness, regular crystal and clear C, Na, Cl, P, and Au signals onto titanium. Further, this composite allowed MC-3T3 E1 growth and viability compared to gelatin and pure titanium. It induced ERK activation and the expression of cell adherent molecules, FAK and SPARC, and growth factor, VEGF. However, GnG decreased the level of SAPK/JNK. This shows that GnG composite coated titanium surfaces have a good biocompatibility for osteoblast growth and attachment than in intact by simple and versatile dipping method. Furthermore, it offers good communication between cell and implant surfaces by regulating cell signaling and adherent molecules, which are useful to enhance the biocompatibility of titanium surfaces. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Titanium as a biomaterial has a strong character cross link with the bone and has been used extensively in dental implants. Titanium shows excellent biocompatibility and high corrosion resistance. However, an unmodified titanium surface is unsuitable for osseointegration [1]. Various surface modification techniques, such as roughness modified, topography, chemistry and electrical charge, etc., have been carried out to improve dental implants [2]. In contrast to physicochemical modification, biological molecules were applied to the surface of an implant to stimulate osteogenic cell growth, proliferation and differentiation [3]. Significant effort has been made to develop an artificial extracellular matrix (ECM) to
∗ Corresponding author at: Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, 634-18, Deokjin-dong, Deokjin-gu, Jeonju, Jeonbuk 561-712, Republic of Korea. Tel.: +82 63 270 44033. E-mail address: [email protected] (H.-K. Yi). 1 These two authors contributed equally to this work. 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.03.069
promote cell growth and differentiation. Among the biomaterials proposed, the composites of hydroxyl apatite (HA) and biodegradable polymers have great potential due to their similar composition to the hard tissue ECM [4]. Gelatin is a natural biodegradable polymer derived from the hydrolysis of collagen [5]. It is completely resorbable in vivo and can imitate the ECM for biocompatibility [6]. In other hand gelatin behaves as stabilizing and reducing agents to prepare gold nano particles. The major advantage for gelatin as a stabilizing agent is that it can be used to tailor the nanocomposite properties and also to provide long-term stability of the nano particles by preventing particles agglomeration [7]. Recently, it was reported that a nano scale or nano structure has a higher biocompatibility than structures on a larger scale [8,9]. Functionalized gold nanoparticles have long been used as tools in bioscience, such as immunostaining marker particles for electron microscopy, chromophores for immune reaction, and nucleic acid hybridization [10–12]. The development of metal nano particles with a well defined shape, size, toxicity, biocompatibility and suitable composition is challenging in the field of nanotechnology. Several methods for the preparation of metal nanoparticles
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to control the size and shapes of particles have been reported [13]. Although, functionalized gold nanoparticles have been used in different phase applications, such as a delivery vehicle, transfection, etc. [14,15]. Less attention has been paid to its use in surface modification in the form of a solid film. It was suggested that the nanoscale modification of titanium implant surface benefits osseointegration and dental implant therapy [16]. Mitogen-activated protein kinase (MAPK) is an essential molecule of the signal transduction machinery and plays a key role in cell growth, differentiation and apoptosis [17]. Other studies have demonstrated that MAPK signaling is involved in osseointegration and osteoblast development on titanium plates [18]. However, there is no report on the relationship between gelatin nano gold (GnG) composite and the MAPK pathway in terms of the regulation of cell adherent expression signaling induced by titanium surfaces. In this study, GnG was selected for the surface modification of titanium and the conditions for cell attachment were examined through the MAPK pathway and cell adherent signal expression. The overall aim of this study was to develop functionalities of GnG on dental implants and fabrication of novel biomaterial. 2. Materials and methods 2.1. Instrumentals The UV–VIS absorption spectra of the samples were recorded on a UV–VIS spectrophotometer (Jasco V-630, Japan). The particle size and morphology were observed by TEM (JEOL JEM 2010) at 200 kV. The surface morphology of the titanium was observed by SEM (SN300 Hitachi Co. Japan). The elemental analysis of the particles was performed using SEM-EDX (JEOL GSM SN-300 Hitachi Co. Japan). 2.2. Materials Gelatin (Type A) from porcine skin, hydrogen tetrachloroaurate (HAuCl4 ) and sodium borohydride (NaBH4 ) were purchased from Sigma Aldrich (St. Louis, MO, USA). All other reagents were obtained from Sigma. Titanium was supplied by Kobe steel (Japan). 2.3. Preparation of gelatin solution and immobilization of gelatin on gold nanoparticles (Gel/Au) 2% gelatin (w/v) was dissolved in distilled water at 4 ◦ C. Gelatin functionalized with gold nanoparticles (Gel/Au) was prepared by an in situ reduction process as described earlier [19]. Briefly, a homogeneous mixture of gelatin (final 1% solutions) and hydrogen tetrachloroaurate (100 M) at a 1:10 ratio was reduced by cold solution of NaBH4 (100 mM). The resulting gelatin stabilized colloids were stored at 4 ◦ C until needed.
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2.4. MC-3T3 E1 cells culture MC-3T3 E1 (mouse osteoblast cell lines) cells were maintained at 37 ◦ C under a humidified, 5% CO2 atmosphere in ␣-MEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 2 mM glutamine, 100 units/ml of penicillin, 100 g/ml of streptomycin, and subcultured at a 1:4 ratio. 2.5. Surface characterization by gelatin and gelatin nano gold composites All titanium foils (grade 2) were cut into pieces (1 cm × 1 cm × 0.1 cm and 6 cm × 6 cm × 0.1 cm), polished with 600 and 1200 grit sandpaper and washed with ultrasonication in water for 10 min. The substrates were then washed with organic solvents (acetone and ethanol) and water in separate ultrasonic baths to remove the residual surface impurities, and blow dried with purified nitrogen gas. Each water soluble 2% gelatin and GnG composite was deposited on a cleaned titanium surface using a dipping technique. In brief gelatin and GnG solution were cast over the titanium samples in a container at 5 min interval and repeated up to 5 times. The samples were removed from the solution, kept at room temperature and dried at ambient condition. The dried coated samples were rinsed with copious amounts of deionized water. The uncoated samples were used as controls. 2.6. Cell viability by trypan blue stain The titanium foils (1 cm × 1 cm × 0.1 cm) in 24-well plates were seeded with 1 × 104 cells and cultured. The cell viability was determined using a trypan blue stain assay. Briefly, the cells were washed twice with PBS, and detached from the titanium surface using Trypsin–EDTA. After all the cells were harvested in PBS, they were stained with 0.2% trypan blue (Trypan Blue stain 0.4%, Gibco BRL, Grand Island, NY, USA). The number of cells was determined under a microscope using a hemocytometer. 2.7. Analysis of cell morphology using crystal violet stain by stereo microscopy The morphology of the cultured cells was determined using a crystal violet stain assay. The titanium foils (1 cm × 1 cm × 0.1 cm) into 24-well plates were seed with 1 × 104 cells and then cultured for the indicated time. For crystal violet staining, the cells were fixed with 2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 15 min at room temperature. After washing with PBS, the cells were incubated with a 0.3% crystal violet solution for 30 min at room temperature. At last, the cells were washed twice with PBS and examined under a stereo microscope (Olympus BX60MF, Japan).
Fig. 1. Appearance and size distribution of gelatin and the GnG composite. (a) The plasmon absorbance of gelatin and the GnG composite. The spectra exhibit absorption bands at approximately 510–530 nm, suggesting the formation of gold nanoparticles. (b) Typical TEM image of the GnG composite. Scale bar represents 20 nm.
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Fig. 2. SEM image of (a) pure titanium (uncoated), (b) titanium surface coated with gelatin and (c) coated with the GnG composite showing rough and nano structures. The image size is 10 m × 10 m.
2.8. Western blot analysis The cells (5 × 106 ) seeded on the titanium surface (6 cm × 6 cm × 0.1 cm) modified with the gelatin and GnG composites were kept into the cell culture dish and normal culture condition for 24 h. The total proteins were extracted with a lysis buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl (pH 8.0), 1%-NP 40, 1 mM aprotinin, 0.1 mM leupeptin, 1 mM pepstatin and quantified using the Bradford dye-binding procedure (Bio-Rad, Hercules, CA, USA). A total 20 g of protein underwent electrophoresis in 8% SDS-polyacrylamide gel under denaturating conditions, and were transferred to a Hybond-P membrane (Amersham, Arlington, IL, USA) using the Mini-protean II system (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk in PBS, the membranes were incubated with the primary antibody. The antibody of phospho-type ERK (#4377) and JNK (#9251) were purchased from Cell signaling (Danvers, MA, USA), and p-FAK (sc-16662), SPARC (sc-25574) and VEGF (sc-507) were purchased from Santa Cruz (Santa Cruz, CA, USA). The actin antibody was obtained from Sigma Aldrich (A-2066, St. Louis, MO, USA). The following conditions were used: a 1:1,000 dilution in either 3% BSA (for ERK and JNK) or 1% skim milk for 24 h at 4 ◦ C. After washing with PBS containing 0.1% Tween-20, once for 15 min and twice for 5 min, the membranes were incubated with secondary antibody, anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase at a 1:3000 dilution in PBS for 1 h at room temperature. After the final wash, the immunoreactive bands were detected on Kodak film by enhanced chemiluminescence.
long time (more than 3 months), as illustrated by the lack of change in the SPB. The titanium surface was modified with gelatin and the GnG composite using dipping methods. Each source material was coated with the composite by dipping into GnG composite and gelatin solution. SEM of pure titanium showed a relatively smooth morphology (Fig. 2a), and the titanium surface coated with gelatin showed a rough and thin layers (Fig. 2b). Wherever, the titanium surface coated with GnG composite showed well distributed crystals of GnG (Fig. 2c). As observed in Fig. 2c, GnG coated surface displayed densely packed structures. Surface characteristics of titanium surface were examined by energy dispersive spectroscopy (EDX). The pure titanium surface showed a clear titanium signal (Fig. 3a). The titanium sample coated with gelatin exhibited a clear Na and Cl signal compared to pure
2.9. Statistical analysis The statistical significance between the groups was assessed using the ANOVA and Ducan’s test. At least three independent experiments were performed. P values < 0.05 were considered significant. 3. Results 3.1. Surface characterization by gelatin and gelatin nano gold composite Gelatin is soluble in water and easily forms a GnG composite. The UV–visible spectra of the GnG composites showed strong absorption at 530 nm, which corresponds to the surface plasmon band (SPB) of gold particles (Fig. 1a), while no absorption was observed for the gelatin solution. In addition, TEM image analysis revealed the detailed features of the GnG composites. The majority of particles had an average diameter of 5 nm and were well dispersed (Fig. 1b). TEM clearly showed that the nanoparticles exhibit a uniform size distribution. Interestingly, the particles were stable for a
Fig. 3. Associated EDX analysis confirming the presence of Au particles in selected titanium surfaces. (a) Pure titanium, (b) titanium surface coated by gelatin and (c) coated by GnG composite.
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Fig. 4. GnG coated titanium surfaces increased cell viability. MC-3T3 E1 cells were seeded onto titanium surfaces coated with 2% gelatin and the GnG composite up to 48 and 72 h. (a) The cell viability was assessed using a trypan blue assay, as described in the materials and methods section. Each value is reported as the mean ± SEM of 3 independent experiments. The cell viability of the GnG coated cells was significantly higher than that of the gelatin coated and control cells (P < 0.05). (b) Stereo microscopy images of the crystal violet stained cell images at the 72 h as described in materials and methods section.
titanium (Fig. 3b). The titanium surface coated with the GnG composite showed signals for Na, Cl, P and Au (Fig. 3c) indicating the formation of a GnG composite and the crystal grown onto the titanium surface. Thus, modification of titanium by GnG composite is easy, simple and a sensibly stable. 3.2. Influence of modified surfaces on MC-3T3 E1 cell biocompatibility The cell viability of the non-modified or modified titanium for MC-3T3 E1 cells was assessed using a trypan blue staining method at different time intervals. The cell viability was higher on the modified titanium specimen than on unmodified one. The GnG composite had a good tendency to increase the cell viability (Fig. 4a). The titanium surface coated with the GnG composite showed cells with good spreading ability and morphology com-
pared with that on the gelatin coated and titanium surface intact (Fig. 4b).An examination of the MAPK signaling pathway showed regulation of the cell survival and death signals. The cells on GnGmodified titanium surface were showed an increase in ERK1/2 activation but a decrease in SAPK/JNK activation compared to pure titanium (Fig. 5a). Similarly, the expression pattern of adherent molecules, such as SPARC, p-FAK and VEGF, was increased. There was greater up regulation of SPARC, p-FAK and VEGF on the GnG composite than on the pure titanium surface (Fig. 5b). 4. Discussion The aim of modifying metal surfaces is to control the tissue–titanium interactions and reduce the bone fixation time [20]. Biological molecules, collagen, and several growth factors have been applied to the surface of implants to improve osseointegration
Fig. 5. Expression of MAPKs and cell adherent molecules on the different titanium surfaces. The MC-3T3 E1 cells were seeded onto pure titanium (control), surfaces coated with gelatin and GnG composite and washed with PBS and the cell culture conditions were maintained as described in materials and methods section. The total cells were harvested after 24 h and assessed by Western blot analysis using specific p-ERK, JNK, SPARC, p-FAK, and VEGF antibodies. To show an equal protein loading, the blots were stripped and incubated with the antibody, actin.
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[21,22]. Gold nano particles are excellent nano templates for the formation of biomimetic materials. In this study, a GnG composite was used to modify of the titanium surface to improve osseointegration, and its biocompatibility was tested using osteoblast cells. The pure titanium and modified titanium surfaces with gelatin or the GnG composite were examined by EDX from specific regions on the titanium surface. The surface of the modified titanium showed stronger C, Na, and Cl signals than pure titanium. In particular, the titanium surface with the GnG composites showed an Au signal. In addition, the GnG composite showed strong absorption in the UV–VIS spectra at 530 nm and had a uniform nanoparticles size according to TEM. These results suggest that the GnG composites formed nanoparticles on nano size. Further, the simple production and well dispersed character of the GnG composite could be applied easily in surface modification and can offer long term stabilization on the titanium. The titanium surface was coated with gelatin and GnG composite. SEM demonstrated that the GnG composite modified surface had good surface roughness. The different surface microtopographies are known to modulate bone cell differentiation and mineralization on titanium implant materials [23]. Therefore, the roughness of the GnG composites on the titanium surface might promote the high activity of the osteoblast attachment. The MC3T3 E1 osteoblast cells showed less viability on pure titanium than on the gelatin modified titanium. Interestingly, the GnG composite on modified titanium surface promoted a good cell spreading ability and higher cell viability than that of gelatin. This is due to the presence of gold nano particles onto the surfaces. Cell adhesion, spreading and migration over the substrate are the first sequential reactions when coming into contact with a material surface, and are essential for cell survival. This study suggests that GnG composites on titanium surfaces creates favorable behaviour for osteoblast survival and results in osseointegration. The MC-3T3 E1 cells on the pure titanium surface showed activation of the SAPK/JNK signal pathway, but not ERK pathways. However, the cells on the titanium surface modified with gelatin and the GnG composite showed inactivation of the JNK pathway and activation of ERK. Interestingly, the cells on the GnG composite showed lower JNK activity and higher ERK activity. It was reported that the attachment of osteoblast cells to the titanium surface is activated by ERK and FAK [24]. In addition, ERK plays a key role in the differentiation, development and growth of osteoblast cells [25]. Therefore, the elevated level of ERK observed on the gelatin and GnG composite suggests that it up-regulates the attachment phase of osteoblast cells and have a more significant role during differentiation and mineralization. The role of SAPK/JNK among the MAPKs in the characterization of osteobalst cells on a titanium surface is unclear. In a similar report, inhibition of the SAPK/JNK pathway led to the inhibition of osteoblast cells apoptosis against heat-shock stress [26]. This suggests that ERK plays a growthrelated function of osteoblast cells on titanium but SAPK/JNK has an opposite action to ERK. So, the lower SAPK/JNK activity on the titanium surface modified with gelatin or GnG composites suggests that the cells have higher resistance to environmental stress than pure titanium, which might lead to an increase in osseointegration. Human hard tissues, such as bones and teeth consist of cells surrounding extracellular matrix. Several proteins are involved in the adhesion to the ECM proteins, cytoskeleton proteins and membrane proteins. In bone, ECM, such as collagen, osteonectin and fibronectin, is essential for mediating cell adhesion to biomaterials, its organization and production modulates the degree of cell attachment to the materials [27]. The mineralized osteoblast ECM is essential for dental implants. Among these, the mineralized osteoblast ECM modulates the secreted proteins that are acidic and rich in cysteine (SPARC or osteonectin) to promote cellular interactions with the extracellular matrix by binding to structural matrix
proteins, such as collagen and vitronectin, and by abrogating focal adhesion, which has a counter adhesive effect on cells [28]. It is a significant bone marker glycoprotein and a modulator of the mineralizing mechanisms that are essential for dental implants [29]. Similarly, focal adhesion kinase (FAK) binds to various structural and signaling proteins that have been implicated in a wide range of cellular processes, including migration, growth factor signaling, cell cycle progression and cell survival [24,30]. Therefore, the elevated levels of SPARC and FAK onto modified titanium surfaces by the GnG composite suggest that modified surfaces assist in the adhesion of osteoblast cells to titanium surface. In addition, vascular epithelial growth factor (VEGF) plays an important role in the regulation of bone remodeling by attracting endothelial cells and osteoblast differentiation [31]. Similarly, VEGF expression of osteoblast cells on the titanium surface suggests its potential role in bone vascularization in the initial phase of wound healing after implant surgery. In this study, gelatin and the GnG composite on the titanium surface had an influence on cell growth. The expression of SPARC and FAK by gelatin and the GnG composite were higher than that on the pure titanium surface. In addition, VEGF was expressed in a similar manner to SPARC and FAK. This shows that modification of the titanium surface with the GnG composite induces the deposition of bone and may increase the life span of an implant. The biocompatibility for biomaterials plays an important role in cellular processing, such as proliferation, differentiation, attachment and protein expression. Accordingly, the expression of SPARC, FAK and VEGF strongly suggest that titanium surfaces modified with GnG composite have higher biocompatibility than other samples. From these results, GnG-modified titanium surface showed increased osteoblast cell viability and ERK activation and decreased SAPK/JNK activation compared to the pure titanium and gelatin coated surface. In addition, modification of the titanium surface induced the expression of cell adherent molecules, such as SPARC, FAK and VEGF. These adherent molecules interact with MAPKs, which is followed by ECM kinase attachment and differentiation of cells on the titanium surface. Furthermore, the inhibition of SAPK/JNK by the GnG composite offers a way of reducing the environment stress on osteoblast cells on a titanium surface. Therefore, the GnG composite on a titanium surface creates favorable conditions for osteoblast attachment and survival. Taken together, the surface modification of titanium with a GnG composite by dipping technique is simpler, advantageous and can be used as a biomaterial and dental implant in various fields of implantation and dentistry. 5. Conclusion This study showed that modification of the titanium surface with GnG composite influences the growth and survival of osteoblast MC-3T3 E1 cells. MAPKs (ERK and SAPK/JNK), cell adherent molecules and kinase (SPARC and FAK) and VEGF illustrated the good biocompatibility of the modified titanium. In particular, this study for the first time showed the relationship between the cell survival, signal pathway and cell adherent molecules in vitro for increasing osseointegration. Overall, the GnG composite provided good conditions for osteoblast cells to grow on the titanium surface through the activation of ERK and FAK, inhibition of SAPK/JNK and the high level of SPARC and VEGF expression, which resulted in MC-3T3 E1 growth and survival. Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. R01-2007-000-20488-0).
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Modified titanium surface with gelatin nano gold composite increases osteoblast cell biocompatibility Young-Hee Lee a,1 , Govinda Bhattarai a,1 , Santosh Aryal b , Nan-Hee Lee a , Min-Ho Lee c , Tae-Gun Kim d , Eun-Chung Jhee a , Hak-Yong Kim b , Ho-Keun Yi a,∗ a
Department of Oral Biochemistry, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju, Republic of Korea Department of Bionanosystem Engineering, Chonbuk National University, Jeonju, Republic of Korea Department of Dental Biomaterials, School of Dentistry and Institute of Oral Bioscience, BK21 program, Chonbuk National University, Jeonju, Republic of Korea d Department of Conservative Dentistry, School of Dentistry, Chonbuk National University, Jeonju, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 27 January 2010 Received in revised form 12 March 2010 Accepted 12 March 2010 Available online 19 March 2010 Keywords: Gelatin nano gold composite Titanium modification Cell signaling Cell adherent molecules Biocompatibility Dental implant
a b s t r a c t This study examined the gelatin nano gold (GnG) composite for surface modification of titanium in addition to insure biocompatibility on dental implants or biomaterials. The GnG composite was constructed by gelatin and hydrogen tetrachloroaurate in presence of reducing agent, sodium borohydrate (NabH4 ). The GnG composite was confirmed by UV–VIS spectroscopy and transmission electron microscopy (TEM). A dipping method was used to modify the titanium surface by GnG composite. Surface was characterized by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). The MC-3T3 E1 cell viability was assessed by trypan blue and the expression of proteins to biocompatibility were analyzed by Western blotting. The GnG composite showed well dispersed character, the strong absorption at 530 nm, roughness, regular crystal and clear C, Na, Cl, P, and Au signals onto titanium. Further, this composite allowed MC-3T3 E1 growth and viability compared to gelatin and pure titanium. It induced ERK activation and the expression of cell adherent molecules, FAK and SPARC, and growth factor, VEGF. However, GnG decreased the level of SAPK/JNK. This shows that GnG composite coated titanium surfaces have a good biocompatibility for osteoblast growth and attachment than in intact by simple and versatile dipping method. Furthermore, it offers good communication between cell and implant surfaces by regulating cell signaling and adherent molecules, which are useful to enhance the biocompatibility of titanium surfaces. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Titanium as a biomaterial has a strong character cross link with the bone and has been used extensively in dental implants. Titanium shows excellent biocompatibility and high corrosion resistance. However, an unmodified titanium surface is unsuitable for osseointegration [1]. Various surface modification techniques, such as roughness modified, topography, chemistry and electrical charge, etc., have been carried out to improve dental implants [2]. In contrast to physicochemical modification, biological molecules were applied to the surface of an implant to stimulate osteogenic cell growth, proliferation and differentiation [3]. Significant effort has been made to develop an artificial extracellular matrix (ECM) to
∗ Corresponding author at: Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, 634-18, Deokjin-dong, Deokjin-gu, Jeonju, Jeonbuk 561-712, Republic of Korea. Tel.: +82 63 270 44033. E-mail address: [email protected] (H.-K. Yi). 1 These two authors contributed equally to this work. 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.03.069
promote cell growth and differentiation. Among the biomaterials proposed, the composites of hydroxyl apatite (HA) and biodegradable polymers have great potential due to their similar composition to the hard tissue ECM [4]. Gelatin is a natural biodegradable polymer derived from the hydrolysis of collagen [5]. It is completely resorbable in vivo and can imitate the ECM for biocompatibility [6]. In other hand gelatin behaves as stabilizing and reducing agents to prepare gold nano particles. The major advantage for gelatin as a stabilizing agent is that it can be used to tailor the nanocomposite properties and also to provide long-term stability of the nano particles by preventing particles agglomeration [7]. Recently, it was reported that a nano scale or nano structure has a higher biocompatibility than structures on a larger scale [8,9]. Functionalized gold nanoparticles have long been used as tools in bioscience, such as immunostaining marker particles for electron microscopy, chromophores for immune reaction, and nucleic acid hybridization [10–12]. The development of metal nano particles with a well defined shape, size, toxicity, biocompatibility and suitable composition is challenging in the field of nanotechnology. Several methods for the preparation of metal nanoparticles
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to control the size and shapes of particles have been reported [13]. Although, functionalized gold nanoparticles have been used in different phase applications, such as a delivery vehicle, transfection, etc. [14,15]. Less attention has been paid to its use in surface modification in the form of a solid film. It was suggested that the nanoscale modification of titanium implant surface benefits osseointegration and dental implant therapy [16]. Mitogen-activated protein kinase (MAPK) is an essential molecule of the signal transduction machinery and plays a key role in cell growth, differentiation and apoptosis [17]. Other studies have demonstrated that MAPK signaling is involved in osseointegration and osteoblast development on titanium plates [18]. However, there is no report on the relationship between gelatin nano gold (GnG) composite and the MAPK pathway in terms of the regulation of cell adherent expression signaling induced by titanium surfaces. In this study, GnG was selected for the surface modification of titanium and the conditions for cell attachment were examined through the MAPK pathway and cell adherent signal expression. The overall aim of this study was to develop functionalities of GnG on dental implants and fabrication of novel biomaterial. 2. Materials and methods 2.1. Instrumentals The UV–VIS absorption spectra of the samples were recorded on a UV–VIS spectrophotometer (Jasco V-630, Japan). The particle size and morphology were observed by TEM (JEOL JEM 2010) at 200 kV. The surface morphology of the titanium was observed by SEM (SN300 Hitachi Co. Japan). The elemental analysis of the particles was performed using SEM-EDX (JEOL GSM SN-300 Hitachi Co. Japan). 2.2. Materials Gelatin (Type A) from porcine skin, hydrogen tetrachloroaurate (HAuCl4 ) and sodium borohydride (NaBH4 ) were purchased from Sigma Aldrich (St. Louis, MO, USA). All other reagents were obtained from Sigma. Titanium was supplied by Kobe steel (Japan). 2.3. Preparation of gelatin solution and immobilization of gelatin on gold nanoparticles (Gel/Au) 2% gelatin (w/v) was dissolved in distilled water at 4 ◦ C. Gelatin functionalized with gold nanoparticles (Gel/Au) was prepared by an in situ reduction process as described earlier [19]. Briefly, a homogeneous mixture of gelatin (final 1% solutions) and hydrogen tetrachloroaurate (100 M) at a 1:10 ratio was reduced by cold solution of NaBH4 (100 mM). The resulting gelatin stabilized colloids were stored at 4 ◦ C until needed.
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2.4. MC-3T3 E1 cells culture MC-3T3 E1 (mouse osteoblast cell lines) cells were maintained at 37 ◦ C under a humidified, 5% CO2 atmosphere in ␣-MEM medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum and 2 mM glutamine, 100 units/ml of penicillin, 100 g/ml of streptomycin, and subcultured at a 1:4 ratio. 2.5. Surface characterization by gelatin and gelatin nano gold composites All titanium foils (grade 2) were cut into pieces (1 cm × 1 cm × 0.1 cm and 6 cm × 6 cm × 0.1 cm), polished with 600 and 1200 grit sandpaper and washed with ultrasonication in water for 10 min. The substrates were then washed with organic solvents (acetone and ethanol) and water in separate ultrasonic baths to remove the residual surface impurities, and blow dried with purified nitrogen gas. Each water soluble 2% gelatin and GnG composite was deposited on a cleaned titanium surface using a dipping technique. In brief gelatin and GnG solution were cast over the titanium samples in a container at 5 min interval and repeated up to 5 times. The samples were removed from the solution, kept at room temperature and dried at ambient condition. The dried coated samples were rinsed with copious amounts of deionized water. The uncoated samples were used as controls. 2.6. Cell viability by trypan blue stain The titanium foils (1 cm × 1 cm × 0.1 cm) in 24-well plates were seeded with 1 × 104 cells and cultured. The cell viability was determined using a trypan blue stain assay. Briefly, the cells were washed twice with PBS, and detached from the titanium surface using Trypsin–EDTA. After all the cells were harvested in PBS, they were stained with 0.2% trypan blue (Trypan Blue stain 0.4%, Gibco BRL, Grand Island, NY, USA). The number of cells was determined under a microscope using a hemocytometer. 2.7. Analysis of cell morphology using crystal violet stain by stereo microscopy The morphology of the cultured cells was determined using a crystal violet stain assay. The titanium foils (1 cm × 1 cm × 0.1 cm) into 24-well plates were seed with 1 × 104 cells and then cultured for the indicated time. For crystal violet staining, the cells were fixed with 2% paraformaldehyde, 0.2% glutaraldehyde in PBS for 15 min at room temperature. After washing with PBS, the cells were incubated with a 0.3% crystal violet solution for 30 min at room temperature. At last, the cells were washed twice with PBS and examined under a stereo microscope (Olympus BX60MF, Japan).
Fig. 1. Appearance and size distribution of gelatin and the GnG composite. (a) The plasmon absorbance of gelatin and the GnG composite. The spectra exhibit absorption bands at approximately 510–530 nm, suggesting the formation of gold nanoparticles. (b) Typical TEM image of the GnG composite. Scale bar represents 20 nm.
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Fig. 2. SEM image of (a) pure titanium (uncoated), (b) titanium surface coated with gelatin and (c) coated with the GnG composite showing rough and nano structures. The image size is 10 m × 10 m.
2.8. Western blot analysis The cells (5 × 106 ) seeded on the titanium surface (6 cm × 6 cm × 0.1 cm) modified with the gelatin and GnG composites were kept into the cell culture dish and normal culture condition for 24 h. The total proteins were extracted with a lysis buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl (pH 8.0), 1%-NP 40, 1 mM aprotinin, 0.1 mM leupeptin, 1 mM pepstatin and quantified using the Bradford dye-binding procedure (Bio-Rad, Hercules, CA, USA). A total 20 g of protein underwent electrophoresis in 8% SDS-polyacrylamide gel under denaturating conditions, and were transferred to a Hybond-P membrane (Amersham, Arlington, IL, USA) using the Mini-protean II system (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk in PBS, the membranes were incubated with the primary antibody. The antibody of phospho-type ERK (#4377) and JNK (#9251) were purchased from Cell signaling (Danvers, MA, USA), and p-FAK (sc-16662), SPARC (sc-25574) and VEGF (sc-507) were purchased from Santa Cruz (Santa Cruz, CA, USA). The actin antibody was obtained from Sigma Aldrich (A-2066, St. Louis, MO, USA). The following conditions were used: a 1:1,000 dilution in either 3% BSA (for ERK and JNK) or 1% skim milk for 24 h at 4 ◦ C. After washing with PBS containing 0.1% Tween-20, once for 15 min and twice for 5 min, the membranes were incubated with secondary antibody, anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase at a 1:3000 dilution in PBS for 1 h at room temperature. After the final wash, the immunoreactive bands were detected on Kodak film by enhanced chemiluminescence.
long time (more than 3 months), as illustrated by the lack of change in the SPB. The titanium surface was modified with gelatin and the GnG composite using dipping methods. Each source material was coated with the composite by dipping into GnG composite and gelatin solution. SEM of pure titanium showed a relatively smooth morphology (Fig. 2a), and the titanium surface coated with gelatin showed a rough and thin layers (Fig. 2b). Wherever, the titanium surface coated with GnG composite showed well distributed crystals of GnG (Fig. 2c). As observed in Fig. 2c, GnG coated surface displayed densely packed structures. Surface characteristics of titanium surface were examined by energy dispersive spectroscopy (EDX). The pure titanium surface showed a clear titanium signal (Fig. 3a). The titanium sample coated with gelatin exhibited a clear Na and Cl signal compared to pure
2.9. Statistical analysis The statistical significance between the groups was assessed using the ANOVA and Ducan’s test. At least three independent experiments were performed. P values < 0.05 were considered significant. 3. Results 3.1. Surface characterization by gelatin and gelatin nano gold composite Gelatin is soluble in water and easily forms a GnG composite. The UV–visible spectra of the GnG composites showed strong absorption at 530 nm, which corresponds to the surface plasmon band (SPB) of gold particles (Fig. 1a), while no absorption was observed for the gelatin solution. In addition, TEM image analysis revealed the detailed features of the GnG composites. The majority of particles had an average diameter of 5 nm and were well dispersed (Fig. 1b). TEM clearly showed that the nanoparticles exhibit a uniform size distribution. Interestingly, the particles were stable for a
Fig. 3. Associated EDX analysis confirming the presence of Au particles in selected titanium surfaces. (a) Pure titanium, (b) titanium surface coated by gelatin and (c) coated by GnG composite.
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Fig. 4. GnG coated titanium surfaces increased cell viability. MC-3T3 E1 cells were seeded onto titanium surfaces coated with 2% gelatin and the GnG composite up to 48 and 72 h. (a) The cell viability was assessed using a trypan blue assay, as described in the materials and methods section. Each value is reported as the mean ± SEM of 3 independent experiments. The cell viability of the GnG coated cells was significantly higher than that of the gelatin coated and control cells (P < 0.05). (b) Stereo microscopy images of the crystal violet stained cell images at the 72 h as described in materials and methods section.
titanium (Fig. 3b). The titanium surface coated with the GnG composite showed signals for Na, Cl, P and Au (Fig. 3c) indicating the formation of a GnG composite and the crystal grown onto the titanium surface. Thus, modification of titanium by GnG composite is easy, simple and a sensibly stable. 3.2. Influence of modified surfaces on MC-3T3 E1 cell biocompatibility The cell viability of the non-modified or modified titanium for MC-3T3 E1 cells was assessed using a trypan blue staining method at different time intervals. The cell viability was higher on the modified titanium specimen than on unmodified one. The GnG composite had a good tendency to increase the cell viability (Fig. 4a). The titanium surface coated with the GnG composite showed cells with good spreading ability and morphology com-
pared with that on the gelatin coated and titanium surface intact (Fig. 4b).An examination of the MAPK signaling pathway showed regulation of the cell survival and death signals. The cells on GnGmodified titanium surface were showed an increase in ERK1/2 activation but a decrease in SAPK/JNK activation compared to pure titanium (Fig. 5a). Similarly, the expression pattern of adherent molecules, such as SPARC, p-FAK and VEGF, was increased. There was greater up regulation of SPARC, p-FAK and VEGF on the GnG composite than on the pure titanium surface (Fig. 5b). 4. Discussion The aim of modifying metal surfaces is to control the tissue–titanium interactions and reduce the bone fixation time [20]. Biological molecules, collagen, and several growth factors have been applied to the surface of implants to improve osseointegration
Fig. 5. Expression of MAPKs and cell adherent molecules on the different titanium surfaces. The MC-3T3 E1 cells were seeded onto pure titanium (control), surfaces coated with gelatin and GnG composite and washed with PBS and the cell culture conditions were maintained as described in materials and methods section. The total cells were harvested after 24 h and assessed by Western blot analysis using specific p-ERK, JNK, SPARC, p-FAK, and VEGF antibodies. To show an equal protein loading, the blots were stripped and incubated with the antibody, actin.
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[21,22]. Gold nano particles are excellent nano templates for the formation of biomimetic materials. In this study, a GnG composite was used to modify of the titanium surface to improve osseointegration, and its biocompatibility was tested using osteoblast cells. The pure titanium and modified titanium surfaces with gelatin or the GnG composite were examined by EDX from specific regions on the titanium surface. The surface of the modified titanium showed stronger C, Na, and Cl signals than pure titanium. In particular, the titanium surface with the GnG composites showed an Au signal. In addition, the GnG composite showed strong absorption in the UV–VIS spectra at 530 nm and had a uniform nanoparticles size according to TEM. These results suggest that the GnG composites formed nanoparticles on nano size. Further, the simple production and well dispersed character of the GnG composite could be applied easily in surface modification and can offer long term stabilization on the titanium. The titanium surface was coated with gelatin and GnG composite. SEM demonstrated that the GnG composite modified surface had good surface roughness. The different surface microtopographies are known to modulate bone cell differentiation and mineralization on titanium implant materials [23]. Therefore, the roughness of the GnG composites on the titanium surface might promote the high activity of the osteoblast attachment. The MC3T3 E1 osteoblast cells showed less viability on pure titanium than on the gelatin modified titanium. Interestingly, the GnG composite on modified titanium surface promoted a good cell spreading ability and higher cell viability than that of gelatin. This is due to the presence of gold nano particles onto the surfaces. Cell adhesion, spreading and migration over the substrate are the first sequential reactions when coming into contact with a material surface, and are essential for cell survival. This study suggests that GnG composites on titanium surfaces creates favorable behaviour for osteoblast survival and results in osseointegration. The MC-3T3 E1 cells on the pure titanium surface showed activation of the SAPK/JNK signal pathway, but not ERK pathways. However, the cells on the titanium surface modified with gelatin and the GnG composite showed inactivation of the JNK pathway and activation of ERK. Interestingly, the cells on the GnG composite showed lower JNK activity and higher ERK activity. It was reported that the attachment of osteoblast cells to the titanium surface is activated by ERK and FAK [24]. In addition, ERK plays a key role in the differentiation, development and growth of osteoblast cells [25]. Therefore, the elevated level of ERK observed on the gelatin and GnG composite suggests that it up-regulates the attachment phase of osteoblast cells and have a more significant role during differentiation and mineralization. The role of SAPK/JNK among the MAPKs in the characterization of osteobalst cells on a titanium surface is unclear. In a similar report, inhibition of the SAPK/JNK pathway led to the inhibition of osteoblast cells apoptosis against heat-shock stress [26]. This suggests that ERK plays a growthrelated function of osteoblast cells on titanium but SAPK/JNK has an opposite action to ERK. So, the lower SAPK/JNK activity on the titanium surface modified with gelatin or GnG composites suggests that the cells have higher resistance to environmental stress than pure titanium, which might lead to an increase in osseointegration. Human hard tissues, such as bones and teeth consist of cells surrounding extracellular matrix. Several proteins are involved in the adhesion to the ECM proteins, cytoskeleton proteins and membrane proteins. In bone, ECM, such as collagen, osteonectin and fibronectin, is essential for mediating cell adhesion to biomaterials, its organization and production modulates the degree of cell attachment to the materials [27]. The mineralized osteoblast ECM is essential for dental implants. Among these, the mineralized osteoblast ECM modulates the secreted proteins that are acidic and rich in cysteine (SPARC or osteonectin) to promote cellular interactions with the extracellular matrix by binding to structural matrix
proteins, such as collagen and vitronectin, and by abrogating focal adhesion, which has a counter adhesive effect on cells [28]. It is a significant bone marker glycoprotein and a modulator of the mineralizing mechanisms that are essential for dental implants [29]. Similarly, focal adhesion kinase (FAK) binds to various structural and signaling proteins that have been implicated in a wide range of cellular processes, including migration, growth factor signaling, cell cycle progression and cell survival [24,30]. Therefore, the elevated levels of SPARC and FAK onto modified titanium surfaces by the GnG composite suggest that modified surfaces assist in the adhesion of osteoblast cells to titanium surface. In addition, vascular epithelial growth factor (VEGF) plays an important role in the regulation of bone remodeling by attracting endothelial cells and osteoblast differentiation [31]. Similarly, VEGF expression of osteoblast cells on the titanium surface suggests its potential role in bone vascularization in the initial phase of wound healing after implant surgery. In this study, gelatin and the GnG composite on the titanium surface had an influence on cell growth. The expression of SPARC and FAK by gelatin and the GnG composite were higher than that on the pure titanium surface. In addition, VEGF was expressed in a similar manner to SPARC and FAK. This shows that modification of the titanium surface with the GnG composite induces the deposition of bone and may increase the life span of an implant. The biocompatibility for biomaterials plays an important role in cellular processing, such as proliferation, differentiation, attachment and protein expression. Accordingly, the expression of SPARC, FAK and VEGF strongly suggest that titanium surfaces modified with GnG composite have higher biocompatibility than other samples. From these results, GnG-modified titanium surface showed increased osteoblast cell viability and ERK activation and decreased SAPK/JNK activation compared to the pure titanium and gelatin coated surface. In addition, modification of the titanium surface induced the expression of cell adherent molecules, such as SPARC, FAK and VEGF. These adherent molecules interact with MAPKs, which is followed by ECM kinase attachment and differentiation of cells on the titanium surface. Furthermore, the inhibition of SAPK/JNK by the GnG composite offers a way of reducing the environment stress on osteoblast cells on a titanium surface. Therefore, the GnG composite on a titanium surface creates favorable conditions for osteoblast attachment and survival. Taken together, the surface modification of titanium with a GnG composite by dipping technique is simpler, advantageous and can be used as a biomaterial and dental implant in various fields of implantation and dentistry. 5. Conclusion This study showed that modification of the titanium surface with GnG composite influences the growth and survival of osteoblast MC-3T3 E1 cells. MAPKs (ERK and SAPK/JNK), cell adherent molecules and kinase (SPARC and FAK) and VEGF illustrated the good biocompatibility of the modified titanium. In particular, this study for the first time showed the relationship between the cell survival, signal pathway and cell adherent molecules in vitro for increasing osseointegration. Overall, the GnG composite provided good conditions for osteoblast cells to grow on the titanium surface through the activation of ERK and FAK, inhibition of SAPK/JNK and the high level of SPARC and VEGF expression, which resulted in MC-3T3 E1 growth and survival. Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (No. R01-2007-000-20488-0).
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